Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells

Abstract

This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37°C allowed the calculation of a kinetic K d of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37°C for 90 min indicated the presence of a single high-affinity site ( K d = 3.87 ± 0.60 nM), saturable and abundant ( B max = 9.64 ± 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1–7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2′-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-( N α-benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 μM. This binding site is, therefore, distinct from angiotensin II receptors, AT 1 and AT 2. Addition of the divalent cations Mg 2+, Mn 2+ or Ca 2+ to the incubation buffer resulted in 90–95% inhibition of the [ 125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites ( B max = 17.8 ± 2.5 pmol/mg protein), with no change in affinity ( K d = 5.7 ± 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTPγS, had no effect on [ 125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.